learning virus genome is fun!

My Biologist and Non-biologist friends, you know, viruses are only microbes, who have either DNA or RNA as genetic material, never both…and for the complex world to be easier, I found a video out there.

you can also check out this awesome site,  http://www.shomusbiology.com/index.html by Suman Bhattacharjee

Hope you enjoy 🙂

also posted at : http://www.sciencenutshell.com/learning-virus-genome-fun/

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Metagenomic virus detection in clinical specimens : a new era

As seen in this Chinese doll picture, the organisms in our nature is not only they look like, they live in an intermingling fashion, which calls for the whole study of them , or the Metagenomics. With the advancement of newer sequencing methods, the area of metagenomics has largely expanded, or on the other way, the broad-range studies of microorganisms in genetic level has geared up the newer and newer inventions of high-throughput sequencing systems. Okay, what it is, Metagenomics is a beautiful study, nothings less important here.

But when we come to the world of viruses, they are not the main ones in the environmental pool. Sure they are present there, but the main sphere of viruses are living cells as they only live well in intracellular condition. In many cases, viruses enter in a body, replicate here, fight with each other or live in peace, spread infections, and when they are shed, from the host body, they are not the same, they have mutated and changed by this time. This made the theory of Quasispecies. So, the environmental study of metagenomics can not be applied here.

Another matter is the studies of Zoonosis. Everyday, there is a new evidence coming out, about, this or that newly emerged disease has come from this or that animals. When a case is found, with no former case, its very difficult to find out the causative agent. So, the specific and effective study of Metagenomics from clinical sample is very important.

This study comes forward with a very new method, tissue-based unbiased virus detection for viral metagenomics (TUViD-VM). Formerly there were many methods available for viral metagenomic study as Hybridization, Sequence independent single primer amplification( SISPA), Arbitrary primed PCR (AP-PCR), Rolling circle amplification etc. This study group from Robert Koch Institute, Germany, not only established a new protocol, but also have shown the efficacy very well.

Schematic description of tissue-based universal virus detection for viral metagenomics protocol. Estimated durations of each step are shown in parentheses. The protocol takes 12 h to complete

This study compared the protocol in different virus groups, different hosts, different extraction, PCR and sequencing methods. The only limitation of this study might be the higher cost for sample processing. It also need high capacity computational analyses as both the viral and host genomes are mixed in a clinical sample gene pool. The researchers nullified the constrain, as sequence data for mammalian hosts are very limited.

This study opened a new era of viral study. Where a newly emerged disease call for an outbreak, and we find nothing in our hands to search the causative agent, as we saw for Ebola just a few days earlier, an established protocol of tissue based viral metagenomics can  help a lot!

Links : http://wwwnc.cdc.gov/eid/article/21/1/14-0766_article

            http://onlinelibrary.wiley.com/doi/10.1002/rmv.532/abstract

also posted at : http://www.sciencenutshell.com/metagenomic-virus-detection-clinical-specimens-new-era/

Next Generation Genome Sequencing – Today And Tommorrow

When Human Genome Project was running, and finally done in 2003, I was in school. The total thing was a sci-fi to me. As they are pouring a drop of blood or a string of hair, and  getting the whole secret story of human written in ATGC ! There were several fun facts published that time, like the distance of sun and earth relative to the length of whole DNA, or the about the mass of it. Sequencing DNA seemed very easy to me until I entered in the Virology lab, where we answer about any unknown thing, like, ahh, do the PCR and sequence it!

In molecular Biology, this thing is common for all organisms, doing PCR and sequencing. The genome is consisted of RNA (only RNA for some viruses) and DNA, but all tends to DNA sequencing, as the RNA is converted into DNA by reverse transcription. DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.

DNA sequencing is largely done by Sanger sequencing along with Maxam-Gilbert sequencing in some little cases. When we talk about sequencing, we usually refer to the chain termination or Sanger sequencing method. We use this in our lab too, for sequencing several parts of viruses to characterize them. I have never done any whole genome sequence, but some here do it by sequencing part by part and then aligning them.

When I came to know about Metagenomics and Next generation Sequencing that sci-fi feeling came back, but in a polished and I-can-do-it version 🙂 So, I decided to share them with my friends, who may know them already. Here I am skipping the whole thing about Sanger method, just adding a schematic picture to differentiate the two generations.

Figure 1.

http://circgenetics.ahajournals.org/content/6/4/427.figures-only

I collected some stuff from Wikipedia and technique types were from just one source, EMBL-EBI website, as anyone can take the online course at a glance. But there are many other methods, maybe I’ll discuss some if the mood comes back 🙂

Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies, which are given below-

Illumina sequencing

Illumina dye sequencing was based on inventions of S Balasubramanian and D Klenerman of Cambridge University. Here, the slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled, with the colour corresponding to the base. They also have a terminator, so that only one base is added at a time. An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added.  The process is repeated, adding one nucleotide at a time and imaging in between. All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out.

http://www.nature.com/nbt/journal/v29/n11/fig_tab/nbt.1996_F1.html

This technique offers a number of advantages over traditional sequencing methods. Due to the automated nature it is possible to sequence multiple strands at once and gain actual sequencing data quickly. Additionally, this method only uses DNA polymerase as opposed to multiple, expensive enzymes required by other sequencing techniques.

454 sequencing

The system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. The DNA fixed to these beads is then amplified by PCR. Each DNA-bound bead is placed into a ~29 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as DNA polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS FLX System for sequencing.

http://www.csulb.edu/~cohlberg/DNAsequencing.html

Ion semiconductor sequencing

Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion. Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. The pH is detected is each of the wells, as each H+ ion released will decrease the pH. The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read.

http://dnamismatch.com/dna-sequencing/next-generation-methods/ion-semiconductor-sequencing/

NGS is significantly cheaper, quicker and is more accurate and reliable than Sanger sequencing. It needs least amount of template DNA, as mainly works on the synthesis process, where Sanger methods depends on chain termination. only one read (maximum ~1kb) can be taken at a time in Sanger sequencing, whereas NGS is massively parallel, allowing 300Gb of DNA to be read on a single run on a single chip. It is also useful for shorter and repeated sequences. Today, Next Generation Sequencing are just outside our lab door, and tomorrow we will slide the door and let it in! 😀

also posted at: http://www.sciencenutshell.com/next-generation-genome-sequencing-today-tommorrow/

Animal Virus 1: Peste Des Petits Ruminants Virus (PPRV)

Dear all, you may find a lot of things at internet about these viruses, but I am feeling at home discussing them, reminding myself the stuffs ..Lets start my Viro blogs with my MS thesis topic, PPRV, which is an animal virus of small animals like goat, sheep, deer and others. It is not associated with big ruminants like cow, buffalo etc. It may not be that concern for rich countries, but a great threat for poorer ones, as they largely depend on domestic farming…Okay, Lets start 🙂

Viral diseases of farm animals are now appearing with regularity in areas where they have never been seen before.  PPRV is currently considered as one of the main animal transboundary diseases that constitutes a threat to livestock production in many developing countries, particularly in western Africa and South Asia. It has been seen in both domestic and wild animals.

The origin of the word “Peste des petits ruminants” is French, meaning “Plague of small ruminants”. It is also known as pseudorinderpest, goat plague, Kata, pneumoenteritis complex, Pest of Small Ruminants, Pest of Sheep and Goats, Stomatitis-Pneumoenteritis Syndrome, goat catarrhal fever etc.

The disease is caused by an RNA virus, Peste des Petits Ruminants virus (PPRV), in the genus Morbillivirus of Paramyxoviridae family. PPRV is antigenically closely related to rinderpest virus (RPV). Other members of the Morbillivirus genus are human measles virus (MV), canine distemper virus (CDV), phocine distemper virus (PDV), dolphin Morbillivirus (DMV) and porpoise Morbillivirus (PMV).

PPRV structure, genome and proteins
PPRV structure, genome and protein
Global distribution of PPRV
Global distribution of PPRV

It is a highly contagious and economically important viral disease of sheep and goats characterized by pyrexia, mucopurulent nasal and ocular discharges, necrotizing and erosive stomatitis, enteritis and pneumonia with very high morbidity . Mortality rate from infection is 10% to 90% depending on age and breed of sheep and goats  which can be as high as 100% .

Infection predominantly spreads via the close contact of the diseased animals which have high fever in the flock with vulnerable animals. The oral, nasal and conjunctival secretions and feces of the infected animals contain a great number of viruses.

The disease was first reported in West Africa during 1942 and later found in Senegal, Central Africa, Sudan, India, East Africa, Arabia, Middle East, Ethiopia, Bangladesh, Pakistan, Nepal, Israel and Saudi Arabia. The disease is now enzootic,  which means “native to”, or “prevalent in”  several African and Asian countries. In 1996, outbreaks of PPRV occurred in the European part of Turkey, and the relevance of these once considered ‘exotic’ viruses is now also high across the European Union and may threaten further regions across the globe in the future.

Antibiotics are not very much effective here, and vaccines are not very much available in poorer countries, mutations in virus is another obstacle. But its close relative virus, Rinderpest is already eradicated from earth (sarcastically which may have helped PPRV reach to more regions and more hosts! ) International organizations  for animal heath are working collectively with locals to eradicate it.

Links:

http://www.oie.int/animal-health-in-the-world/official-disease-status/peste-des-petits-ruminants/

http://www.fao.org/docrep/003/X1703E/X1703E00.htm

http://wwwnc.cdc.gov/eid/article/20/12/14-0684_article

Amphibian me!

When I decided to write about the second assignment on the theme and tagline, it was night already, and was so tired. When I woke up, another topic was already published! It was, say hello to neighbors ! I thought to write on both in one piece, and its night already 😀

As I said it earlier, my English is quite clumsy, as it wasn’t my first language…I didn’t read much English literature as well. But these days, I wanted to fly in  different skies, meet new birds, and English gives nice wings for that! That’s why, I am here!

My tagline, ” stories of mountains is history, stories of pebbles is life”- came from an inspiration. I am reading a book these days, “Love in the land of Midas” by Kapka Kassakova, there is a line, “These are the forces of history and I’m just a pebble tumbling down the hill before the great avalanche” . Yes we all are pebbles here, we won’t be in the history, so what? let tell our stories ourselves! Probably, that’s why Literature was born 🙂

Oh my, joining the neighbors! That took the soul out of me ! It was harder than our neighbor aunty’s, “could you give me some this? or that? or the whole!” When I searched for topics, I was scared, what am I doing? I was trying to hide myself from science, take a breath in history, novels, mythology etc, but I was searching topics like microbiology, cell biology, virology…In a second again arts ones…Started following all, and soon my reader section was a mess!! what should I read and what not!! Feeling like a frog, amphibian one, swimming in arts, and staying in a science hole! So, I put my original soul in “about.me” section 😀

I found lots of people here today, who are just like me, rather they are, Jack of all, and master of all! so, I started thinking of  writing about both…But I am very confused in this matter…After a blog on streets, or street people, suddenly stumbling in a blog like Protein synthesis or some virus, how would it look like?? Again, anyone can find them easily on internet, so should I write them in a blog?  If I do, should I write a new blog about that? But it’ll be very difficult for me to run them two…now, what should I do??